The invention relates to (androst-5-en-17xcex2-yl)alkyl sulfoxides and sulfones, to pharmaceutical compositions containing the same, as well as to the use: of these (androst-5-en-17xcex2-yl)alkyl sulfoxides and sulfones for the preparation of a medicament for the control of fertility.
Sexual reproduction involves a cyclic alternation of diploid and haploid states: diploid cells divide by the process of meiosis to form haploid cells, and the haploid cells fuse in pairs at fertilization to form new diploid cells. The process of meiosis is characterized by two meiotic divisions, unique to both male and female germ cells. During the process two cell divisions, following one round of DNA replication, give rise to four haploid cells from one single diploid cell. Chromosomal crossover events, during which paternal and maternal genetic material is exchanged, occur during the prophase of the first meiotic division. At the end of the first meiotic division one member of each chromosome pair, composed of two sister chromatids is distributed to each daughter cell. The second meiotic division segregates each sister chromatide into a separate haploid cell. Male and female germ cells are subject to similar meiotic divisions but differ in the regulation of these processes. In the male meiosis is a continuous process in germ cells derived from a population of immature germ cells, the stem cell spermatogonia. After sexual maturation of the male, spermatogonia from this stem cell population embark on meiosis. The first and second meiotic division proceed without interruption and eventually give rise to four mature spermatozoa.
In the female, primary oocytes start the first meiotic division already during the embryonic stage but they remain arrested in the prophase (dictyate stage) until the female becomes sexually mature. Meiosis resumes at the time of ovulation (egg maturation) after which the first meiotic division is completed and the second meiotic division is initiated. In most vertebrates the second meiotic division is arrested at the metaphase and only completed after fertilization. At the end of the first and of the second meiotic division the cytoplasm divides asymmetrically to produce two secondary oocytes, each with a haploid number of single chromosomes, but greatly differing in size: one is a small polar body, which eventually degenerates, and the other is a large cell containing all the developmental potential. Finally one mature ovum is produced.
The stage at which the developing oocyte is released from the ovary and is ready for fertilization differs in different species. In both invertebrates and vertebrates ovarian accessory cells respond to polypeptides (gonadotropins) produced elsewhere in the body so as to control the maturation of the oocyte and eventually (in most species) ovulation. In humans the primary oocytes of the newborn female are arrested in prophase of meiotic division I and most are surrounded by a single layer of follicle cells; such an oocyte with its surrounding cells constitute the primordial follicle. A small portion of primordial follicles sequentially begins to grow to become developing follicles: the follicle cells enlarge and proliferate to form a multilayered envelope around the primary oocyte; the oocyte itself enlarges and develops the zona pellucida, an extracellular matrix consisting largely of glycoproteins, and cortical granules, specialized secretory vesicles just under the plasma membrane in the outer region, the cortex, of the egg cytoplasm [when the egg is activated by a sperm, these cortical granules release their contents by exocytosis; the contents of the granules act to alter the egg coat so as to prevent other sperms from fusing with the egg].
The developing follicles grow continuously and some of them develop a fluid-filled cavity, or antrum, to become antral follicles. Development of such follicles is dependent on gonadotropins (mainly follicle stimulating hormone xe2x88x92FSH) secreted by the pituitary gland and on estrogens secreted by the follicle cells themselves. Starting at puberty, a surge of secretion by the pituitary of another gonadotropin, luteinizing hormone (LH), activates a single antral follicle to complete its development: the enclosed primary oocyte matures to complete the meiotic division I as the stimulated follicle rapidly enlarges and ruptures at the surface of the ovary, releasing the secondary oocyte within. As is the case with most mammals, the secondary oocyte is triggered to undergo division II of meiosis only if it is fertilized by a sperm.
Studies on the mechanisms controlling initiation and regulation of the meiotic process in male and female germ cells suggest a role for cyclic nucleotides in mediating meiotic arrest. Spontaneous maturation of oocytes can be prevented by compounds that maintain elevated cAMP levels [Eppig, J. and Downs, S. (1984) Biol. Reprod. 30: 1-11]. Purines, like adenosine or hypoxanthine, are thought to be involved in the cAMP mediated maintenance of meiotic arrest [Eppig, J., Ward-Bailey, P. and Coleman, D. (1985) Biol. Reprod. 33: 1041-1049]. The presence of a meiosis regulating substance in a culture system of fetal mouse gonads was first described by Byskov, A. et al (1976) Dev. Biol. 52: 193-200. It was suggested that the concentrations of a meiosis activating substance (MAS) and a meiosis preventing substance (MPS) regulate the meiotic process in concert [Byskov, A. et al. (1994). In xe2x80x9cThe physiology of reproductionxe2x80x9d, Eds. Knobil, E. and Neill, J., Raven Press, New York]. More recently (3xcex2,5xcex2,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol (FF-MAS), isolated from human follicular fluid, and (3xcex2,5xcex1,20R)-4,4-dimethyl-cholesta-8,24-dien-3-ol, isolated from bull testes, were identified by Byskov, A. et al [(1995), Nature 374: 559-562] as endogenous meiosis activating substances in human and bovine, respectively. These sterols proved to be able to activate the resumption of meiosis in cultured cumulus enclosed and naked mouse oocytes.
Derivatives of the endogenous sterols, having either a saturated or an unsaturated cholestane side chain, have been disclosed in the international patent application WO98/28323 (NOVO NORDISK A/S) as meiosis inhibiting substances. Meiosis inhibiting substances are compounds that are antagonists of a naturally occurring meiosis activating substance. Thus, they could be used for contraception. A drawback of the compounds described in this patent application is that they are prone to rapid deactivation in the body [Hall, P. F. (1985) Vitamins and Hormones, 42: 315], thereby restricting their therapeutic potential as fertility control agents.
A need therefore exists for inhibitors of the meiotic process having improved in vivo activity.
To this end, the invention provides a group of compounds which has been found to possess this quality. It has thus been found that certain (androst-5-en-17xcex2-yl)alkyl sulfoxides and sulfones show improved meiosis inhibiting activity.
Accordingly, the invention resides in the use of (androst-5-en-17xcex2-yl)alkyl sulfoxides and sulfones having the general formula I given below, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, for manufacturing a medicament for the control of fertility. 
wherein
R1 is (H,OR), (H,OSO3H) or NOR; with R being H, (C1-6)alkyl or (C1-6)acyl;
each of R2 and R3 is independently hydrogen or (C1-6)alkyl;
n is 0, 1, 2 or 3;
X is S(O) or S(O)2;
Y is xe2x80x94CH2xe2x80x94CR4xe2x95x90CR5R6, wherein each of R4, R5 and R6 is independently hydrogen,
phenyl, halogen or (C1-4)alkyl, optionally substituted by hydroxy, (C1-4)alkoxy, oxo or halogen; or R4 and R5 together with the carbon atoms to which they are bound form a (C5-6)cycloalkene ring; or R5 and R6 together with the carbon atom to which they are bound form a (C3-6)cycloalkane ring; or
Y is xe2x80x94CHxe2x95x90CR7xe2x80x94CR8R8R10, wherein each of R7, R8 and R9 is independently hydrogen, phenyl or (C1-4)alkyl, optionally substituted by hydroxy, (C1-4)alkoxy, oxo or halogen; R10 is hydrogen, hydroxy or fluorine; or R7 and R8 together with the carbon atoms to which they are bound form a (C3-6)cycloalkane ring; or R8 and R9 together with the carbon atoms to which they are bound form a (C3-6)cycloalkane ring; or
Y is xe2x80x94CH2xe2x80x94Cxe2x89xa1CR11, wherein R11 is hydrogen or (C1-4)alkyl, optionally substituted by hydroxy, (C1-4)alkoxy, oxo or halogen; or R11, is phenyl, optionally substituted by hydroxy, (C1-4)alkoxy, (C1-4)alkyl or halogen; or
Y is phenyl, optionally substituted by hydroxy, (C1-4)alkoxy, (C1-4)alkyl or halogen; and the dotted line indicates an optional additional bond.
The invention also resides in a method of treatment, comprising the administration to a human female of child-bearing age, or to a fertile human male, of a medicament for the control of fertility, wherein said medicament comprises a (androst-5-en-17xcex2-yl)alkyl sulfoxide or sulfone having the above general formula I (or a produg thereof or a pharmaceutically acceptable salt thereof).
The compounds used according to this invention have not been known before as having meiosis-inhibiting activity, or for any other capacity indicating their usefulness in the control of fertility. Many of these compounds being novel per se, the invention also resides in the above compounds, i.e. (androst-5-en-17xcex2-yl)alkyl sulfoxides and sulfones having the above formula I or a pharmaceutically acceptable salt thereof, with the proviso that those compounds which incidentally have been known are excluded, said compounds being:
(3xcex2,20S)-20-methyl-21-(phenylsulfonyl)pregna-5,7-dien-3-ol, which is known from DD 247008, and DD 282230;
(3xcex2,20S)-20-methyl-21-(phenylsulfonyl)pregna-5,7-dien-3-ol acetate, which is known from DD 272854 and DD 282230;
(3xcex2,20S)-20-methyl-21-(phenylsulfonyl)pregna-5,7-dien-3-ol benzoate, which is known from DD 282230;
[3xcex2,20S,(21S)]-4,4,20-trimethyl-21-[(2-methyl-2-propenyl)sulfinyl]pregn-5-en-3-ol which is known as a by-product from WO 98/55498.
The invention further resides in a pharmaceutical composition comprising a (androst-5-en-17xcex2-yl)alkyl sulfoxide or sulfone having the general formula I. Pharmaceutical compositions which comprise the disclaimers are within the ambit of the present invention.
The term (C1-6)alkyl as used in the definition of formula I means a branched or unbranched alkyl group having 1-6 carbon atoms, like hexyl, pentyl, butyl, isobutyl, tertiary butyl, propyl, isopropyl, ethyl and methyl. Likewise, the term (C1-4)alkyl means an alkyl group having 1-4 carbon atoms.
The term (C3-6)cycloalkane ring means a cycloalkane ring having 3-6 carbon atoms, like cyclopropane, cyclobutane, cyclopentane and cyclohexane.
The term (C5-6)cycloalkene ring means a cycloalkene ring having at least one double bond and 5 or 6 carbon atoms.
The term (C1-6)acyl means an acyl group derived from a carboxylic acid having from 1-6 carbon atoms, like hexanoyl, pentanoyl, pivaloyl, butyryl, propanoyl, acetyl and formyl. Also included within the definition of (C1-6)acyl are acyl groups derived from dicarboxylic acids, like hemi-glutaroyl, hemi-succinoyl, and hemi-maloyl. A preferred (C1-6)acyl group is hemi-succinoyl.
The term (C1-4)alkoxy means an alkyloxy having 1-4 carbon atoms, like butyloxy, propyloxy, isopropyloxy, ethyloxy, and, preferably, methyloxy.
The term halogen means F, Cl, Br or I. When halogen is a substituent at an alkyl group, like in the definition of R4-9 and R11, Cl and F are preferred, F being most preferred.
It is understood that the (androst-5-en-17xcex2-yl)alkyl sulfoxides and sulfones of the invention have the natural configurations 8xcex2, 9xcex1, 10xcex2, 13xcex2, 14xcex1, 17xcex2. Preferred compounds according to the invention are the (androst-5-en-17xcex2-yl)alkyl sulfoxides and sulfones of formula I wherein n is 0 or 1. More preferred are the comrpounds wherein in addition R1 is (H,OR), wherein R has the previously given meaning, and the dotted line indicates a saturated bond. Among these preferred compounds those with the 3-OR substituent in the xcex2-configuration are especially preferred. The configuration at position 20 of the (androst-5-en-17xcex2-yl)alkyl sulfoxides and sulfones of the invention can be either R or S. Specifically preferred compounds of the invention are [3xcex2,20S,(21R)]-4,4,20-trimethyl-21-[(2-methyl-2-propenyl) sulfinyl]pregn-5-en-3-ol and [3xcex2,20S,(21R]-4,4,20-trimethyl-21 -(phenylsulfinyl) pregn-5-en-3-ol.
The meiosis inhibiting activity of the (androst-5-en-17xcex2-yl)alkyl sulfoxides and sulfones of the invention is measured in an in vitro oocyte assay as the ability to inhibit the FF-Mas or 22S-hydroxy-FF-Mas induced maturation, in hypoxanthine media, in denuded oocytes (DO) or cumulus enclosed oocytes (CEO), respectively. The compounds can be used to inhibit meiosis in both male and female and thus can be used as contraceptive agents.
(Androst-5-en-17xcex2-yl)alkyl sulfoxides and sulfones of the invention can be used for female contraception by inhibition of the naturally induced oocyte mraturation a caused by the gonadotrophin surge. These compounds lead to the ovulation of an immature oocyte which cannot be fertilized.
For male contraception the compounds of the invention can be administered to inhibit the spermatogenesis.
The (androst-5-en-17xcex2-yl)alkyl sulfoxides and sulfones of this invention have the natural configurations 8xcex2, 9xcex1, 10xcex2, 13xcex2, 14xcex1, 17xcex2, and possess also one or more additional chiral carbon atoms. The compounds may therefore be obtained as a pure diastereomer, or as a mixture of diastereomers. Methods for obtaining the pure diastereomers are well known in the art, e.g. crystallization or chromatography.
Compounds according to formula I wherein X is S(O) may exist as a diastereoisomeric sulfoxide pair, due to the presence of the optically active sulfur atom. Both the diastereoisomeric mixture and the separate isomers are included in the present invention.
For therapeutic use, salts of the compounds of formula I are those wherein the counterion is pharmaceutically acceptable. However, salts of the acids according to formula I [i.e. compounds wherein R1 is (H,OSO3H)] may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound. All salts, whether pharmaceutically acceptable or not, are included within the ambit of the present invention. Examples of salts of acids according to the invention are mineral salts such as sodium salt, potassium salt, and salts derived from organic bases like ammonia, imidazole, ethylenediamine, triethylamine and the like.
The compounds of formula I or a pharmaceutically acceptable salt thereof, also referred to herein as the active ingredient, may be administered enterally or parenterally. The exact dose and regimen of administration of the active ingredient, or a pharmaceutical composition thereof, will necessarily be dependent upon the therapeutic effect to be achieved (e.g. female contraception or male contraception), and will vary with the particular compound, the route of administration, and the age and condition of the individual subject to whom the medicament is to be administered.
In general parenteral administration requires lower dosages than other methods of administration which are more dependent upon adsorption. However, a dosage for humans preferably contains 0.0001-25 mg per kg body weight. The desired dose may be presented as one dose or as multiple subdoses administered at appropriate intervals throughout the day, or, in case of female recipients, as doses to be administered at appropriate daily intervals throughout the menstrual cycle. The dosage as well as the regimen of administration may differ between a female and a male recipient.
In case of in vitro or ex vivo applications,the compounds of the inventions are to be used in the incubation media in a concentration of approximately 0.01-5 xcexcg/ml.
The present invention thus also relates to pharmaceutical compositions comprising a (androst-5-en-17xcex2-yl)alkyl sulfoxide or sulfone according to formula I in admixture with pharmaceutically acceptable auxiliaries, and optionally other therapeutic agents. The auxilliaries must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof.
Pharmaceutical compositions include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may be prepared by any method well known in the art of pharmacy, for example, using methods such as those described in Gennaro et al., Remington""s Pharmaceutical Sciences (18th ed., Mack Publishing Company, 1990, see especially Part 8: Pharmaceutical Preparations and Their Manufacture). Such methods include the step of bringing in association the active ingredient with any auxilliary agent. The auxilliary agent(s), also named accessory ingredients, include those conventional in the art (Gennaro, supra), such as, fillers, binders, diluents, disintegrants, lubricants, colorants, flavoring agents and wetting agents.
Pharmaceutical compositions suitable for oral administration may be presented as discrete dosage units such as pills, tablets or capsules, or as a powder or granules, or as a solution or suspension. The active ingredient may also be presented as a bolus or paste. The compositions can further be processed into a suppository or enema for rectal administration.
For parenteral administration, suitable compositions include aqueous and non-aqueous sterile injection. The compositions may be presented in unit-dose or multi-dose containers, for example sealed vials and ampoules, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of sterile liquid carrier, for example, water prior to use.
Compositions, or formulations, suitable for administration by nasal inhalation include fine dusts or mists which may be generated by means of metered dose pressurised aerosols, nebulisers or insufflators.
The (androst-5-en-17xcex2-yl)alkyl sulfoxides and sulfones of the invention can also be administered in the form of implantable pharmaceutical devices, consisting of a core of active material, encased by a release rate-regulating membrane. Such implants are to be applied subcutaneously or locally, and will release the active ingredient at an approximately constant rate over relatively large periods of time, for instance from weeks to years. Methods for the preparation of implantable pharmaceutical devices as such are known in the art, for example as described in European Patent 0,303,306 (AKZO N.V.).
The compounds of the invention may be produced by various methods known in the art of organic chemistry in general, and especially in the art of the chemistry of steroids (see, for example: Fried, J. and Edwards, J. A., xe2x80x9cOrganic Reactions in Steroid Chemistryxe2x80x9d, Volumes I and II, Van Nostrand Reinhold Company, New York, 1972). A suitable method for the preparation of compounds of formula I is the reaction of a compound of general formula II, 
wherein R2 and R3 have the previously given meaning, R12 is a hydroxy-protecting group such as an acyl group, like an acetyl group, a benzoyl group or a pivaloyl group, or an alkoxyalkyl group, like an ethoxyethyl group or a tetrahydropyranyl (THP) group, n is 0, 1, 2 or 3, and wherein Z is a halogen, preferably bromide or iodide, or a leaving group like tosyloxy or mesyloxy, and the dotted line indicates an optional bond, with a 2-propenyl mercaptan, a 2-propynyl mercaptan or a thiophenol derivative, or the reaction of a compound according to formula II wherein Z is SH, with a 2-propenyl chloride, bromide or iodide, or a 2-propynyl chloride, bromide or iodide, or with a 2-propenyl or 2-propynyl alcohol derivative wherein the hydroxy group is converted to a leaving group, such as mesyloxy or tosyloxy,
whereafter a 17xcex2-aryl(2-propenyl)(2-propynyl)thioalkyl-androst-5-ene derivative is oxidized to the corresponding sulfoxide [X=S(O)] or sulfone [X=S(O)2] derivative, whereafter a 17xcex2-(2-propenylsulfinyl)alkyl-androst-5-ene derivative or 17xcex2-(2-propenylsulfonyl)alkyl-androst-5-ene derivative is optionally isomerized to the 17xcex2-(1-propenylsulfinyl)alkyl-androst-5-ene derivative or 17xcex2-(1-propenylsulfonyl)alkyl-androst-5-ene derivative, respectively, after which any remaining protecting groups are removed, and the 3-OH group of the product is optionally converted to a 3-OR group, wherein R has the meaning as previously defined, or wherein the 3-OH group is oxidized and the resulting 3-keto-group converted to NOR, wherein R has the meaning as previously defined.
The preparation of compounds having formula 11 is carried out using methods known in the art.
For instance, compounds of formula II (Z=OH, R12=acyl) can be obtained from compounds having formula II (Z=OSit-BuMe2, R12=acyl), described in WO-09852965 and WO-09855498, by hydrolysis of the silyl ether.
Compounds of formula II (Z=OH) in which the 3-hydroxy function is protected as an alkoxyalkyl ether can be obtained from compounds of formula II (Z=OSit-BuMe2, R12=acyl) as follows: the ester function at C-3 is removed by reduction with lithium aluminium hydride, or by other hydride reducing agents known in the art, whereafter the 3-hydroxy group is reprotected as an alkoxyalkyl ether, e.g. an ethoxyethyl ether or a tetrahydropyranyl ether. Finally, treatment with a fluoride agent, e.g. potassium fluoride, tetrabutylammonium fluoride or other reagents known in the art then results in the formation of the unsaturated diol derivatives of formula II (Z=OH) in which the 3-hydroxy function is protected as an alkoxyalkyl ether. These and other suitable protective groups are known in the art, e.g. from Greene, T. W. and Wuts, P. G. M.: Protective groups in Organic Synthesis, Wiley, N.Y., 1991.
Compounds having formula II (Z=SH) are obtained from compounds of formula II (Z=OH) using methods known in the art (see e.g. Schoberl, A. et al, Methoden der Organischen Chemie (Houben-Weyl), Band IX, p. 7, Georg Thieme Publishers, Stuttgart, 1955). For instance, this conversion can be carried out by means of a Mitsunobu reaction with thioacetic acid followed by conversion to the thiol derivative by reduction or saponification [see Hughes, D. L., Organic Reactions 42, 335 (1992)]. Alternatively, they can be prepared by reaction of compounds of general formula II in which Z is a leaving group such as bromide, iodide, tosyloxy and mesyloxy, with thiourea followed by reaction with a base, like sodium hydroxide or potassium hydroxide [see e.g. Allewaert, K. et al, Bioorganic and Med. Chem. Lett. 3, 1859 (1993)].
The etherification reaction of compounds having formula II is carried out using methods known in the art.
For instance, compounds of formula I (X=S; Y=aryl, 2-propenyl, 2-propynyl) can be prepared by reaction of compounds of general formula II, wherein Z is a leaving group such as bromide, iodide, tosyloxy and mesyloxy, with a suitably substituted 2-propenyl mercaptan derivative, a 2-propynyl mercaptan derivative, or a thiophenol derivative, which is previously converted to the lithium, sodium or potassium salt [see e.g. Meerwein, H., Methoden der Organischen Chemie (Houben-Weyl), Band VI/3, p. 1, Georg Thieme Publishers, Stuttgart, 1965; Yin, J. et al, Tetrahedron Lett. 38, 5953 (1997) and references cited therein].
Compounds of formula I (X=S; Y=2-propenyl, 2-propynyl) can also be prepared analogously by means of an etherification reaction between a compound of formula II wherein Z is SH, which is previously converted to the lithium, sodium or potassium salt, with a suitably substituted 2-propenyl or 2-propynyl halide (Cl,Br,I) or with a suitably substituted 2-propenyl or 2-propynyl alcohol derivative in which the hydroxy group is converted to a leaving group, such as mesyloxy or tosyloxy.
Compounds of general formula I wherein X is S(O) or S(O)2 can be prepared from compounds of formula I wherein X is S by oxidation using agents capable of converting a thio ether into a sulfoxide or a sulfone, e.g. sodium periodate, oxone, H2O2 and others [see e.g. Varma, R. S. et al, Tetrahedron Lett. 38, 6525 (1997) and references cited therein].
The isomerization of 17xcex2-(2-propenylsulfinyl)alkyl-androst-5-ene derivatives or 17xcex2-(2-propenylsulfonyl)alkyl-androst-5-ene derivatives to the 1-propenyl derivatives can be carried out with base, e.g. potassium tert-butoxide, sodium ethoxide, and others [see e.g. Svata, V. et al, Collect. Czech. Chem. Commun. 2619 (1978); Kimmelma, R., Acta Chem. Scand. 47, 1201 (1993); Maercker, A. Et al, J. Organomet. Chem. 116,21 (1976)].
Compounds of general formula I [X=S(O)2; n=1,2,3] can also be prepared directly by reaction of compounds of general formula II in which Z is a leaving group such as bromide, iodide, tosyloxy and mesyloxy, with an unsubstituted or suitably substituted benzenesulfinic acid derivative, which is previously converted to the sodium or potassium salt [see Trost, B. M. et al, J. Am. Chem. Soc. 105, 5075 (1983)].
Compounds of formula I in which R1 is (H,OH) may serve as starting material for the synthesis, using methods known in the art, of compounds of formula I in which R1 is (H,OR), (H,OSO3H) or NOR, and R is H, (C1-6)alkyl, or (C1-6)acyl.